This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

Inadequate clearance of apoptotic cells by macrophages is one of the reasons for the
breakdown of self-tolerance. Class A scavenger receptors, macrophage receptor with
collagenous structure (MARCO) and scavenger receptor A (SR-A), which are expressed
on macrophages, play important roles in the uptake of apoptotic cells. A previous
study reported the presence of the anti-MARCO antibody in lupus-prone mice and systemic
lupus erythematosus (SLE) patients. The purpose of this study was to investigate the
prevalence of anti-class A scavenger receptor antibodies in patients with various
autoimmune diseases, in particular SLE, and the functional implication of those autoantibodies
in the phagocytic clearance of apoptotic cells by macrophages.

Methods

Purified recombinant scavenger receptor cysteine-rich (SRCR) polypeptide (ligand-binding
domain of MARCO) and recombinant SR-A were used as antigens. By using enzyme-linked
immunosorbent assay, the anti-SRCR and anti-SR-A antibodies were detected in the sera
of untreated patients with SLE (n = 65), rheumatoid arthritis (n = 65), primary Sjögren
syndrome (n = 25), and healthy blood donors (n = 85). The effect of IgG purified from
SLE patients or healthy controls on the phagocytosis of apoptotic cells by macrophages
was measured by the flow cytometry assay.

Results

Anti-SRCR antibodies were present in patients with SLE (18.5%) and rheumatoid arthritis
(3.1%), but not in those with primary Sjögren syndrome. Anti-SR-A antibodies were
present in patients with SLE (33.8%), rheumatoid arthritis (13.8%), and primary Sjögren
syndrome (12.0%). IgG from SLE patients positive for anti-SRCR or anti-SR-A antibodies
showed a higher inhibition rate on binding of apoptotic cells to macrophages than
IgG from healthy controls (both P < 0.05). IgG from SLE patients positive for both anti-SRCR and anti-SR-A antibodies
showed a significantly higher inhibition rate on ingestion of apoptotic by macrophages
than IgG from healthy controls (P < 0.05).

Conclusions

Our results indicated that autoantibodies to class A scavenger receptors might contribute
to the breakdown of self-tolerance by impairing the clearance of apoptotic debris
and play a role in the pathogenesis of autoimmune disease, especially in SLE.

Introduction

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized
by the production of a wide range of autoantibodies. Several lines of evidence suggest
that increased apoptosis and impaired phagocytic clearance of apoptotic cells could
play important roles in the breakdown of self-tolerance because they lead to autoantigen
overload, a decrease in anti-inflammatory cytokine production, and in susceptible
individuals, initiation of an autoimmune response [1]. Studies on human SLE have shown increased apoptosis of peripheral blood mononuclear
cells, neutrophils, and macrophages [2,3]. In addition to aberrant apoptosis, macrophages from patients with SLE exhibited
impaired clearance of apoptotic cells both in vitro and in vivo [3-6].

However, it is unclear whether macrophages of patients with SLE have intrinsic defects
resulting in reduced clearance of apoptotic cells; serum factors have been implicated
in the reduced clearance of apoptotic cells by macrophages. The complex process of
phagocytosis involves a range of receptors, ligands, and opsonins, which are involved
in the recognition and internalization of apoptotic cells. Recent studies indicated
that class A scavenger receptor member macrophage receptor with collagenous structure
(MARCO) and scavenger receptor A (SR-A) could bind to apoptotic cells and contribute
to the clearance of apoptotic cells [7,8]. Interestingly, subsequent study by Wermeling et al. [8] showed that FcγRIIB-/- (NZB × NZW) F1 mice, which develop spontaneous SLE, produce autoantibodies that are
capable of recognizing MARCO and SR-A. Study has also shown that patients with SLE
also produce autoantibodies to MARCO. It is proposed that autoantibodies blocking
scavenger receptors may alter the phagocytosis of apoptotic cells by macrophages,
and, thereby, facilitate the development of an autoimmune response.

Class A scavenger receptors appear to be new target antigens in SLE. MARCO is a trimeric
membrane protein containing a short N-terminal intracellular domain, a transmembrane
domain, and a large extracellular domain composed of a spacer domain, a long collagenous
domain, and a C-terminal scavenger receptor cysteine-rich (SRCR) domain. SRCR plays
a major role in the ligand-binding function of MARCO [9,10]. The binding of autoantibodies to MARCO in (NZB × NZW) F1 mice could be blocked by
an antibody to SRCR [8], implicating SRCR as a potential autoantigen in SLE. SR-A, the other class A scavenger
receptor member that is also capable of binding with apoptotic cells, is structurally
quite similar to MARCO; however, it differs from MARCO in that it has an a-helical
coiled-coil domain, but a short collagenous domain and its ligand-binding function
have been localized to the collagenous domain. Considering the possibility that SRCR
and SR-A could serve as autoantigens in autoimmune diseases, we evaluated the prevalence
of anti-SRCR antibodies and anti-SR-A antibodies in patients with SLE and other various
systemic autoimmune diseases. Furthermore, we investigated whether these autoantibodies
could interfere with the phagocytic clearance of apoptotic cells by macrophages.

Materials and methods

Patients and healthy controls

All patients in this study were followed at the Renji Hospital affiliated with Shanghai
Jiaotong University School of Medicine, Shanghai, China. The consecutively recruited
patients include 65 untreated new onset patients with SLE who fulfilled the American
College of Rheumatology (ACR) classification criteria for the diagnosis of SLE [11], 65 patients with rheumatoid arthritis (RA) who satisfied the ACR criteria for RA
[12], 25 patients with primary Sjögren syndrome (pSS) diagnosed according to the American-European
consensus group criteria [13], and 85 healthy blood donors. Demographic data were collected for all patients (expect
for RA patients) and have been presented in Table 1. All the serum samples were stored at -80°C. Disease activity was measured using
the SLE disease activity index 2000 (SLEDAI-2K) [14]. The main clinical and serological features of SLE patients are shown in Additional
file 1. The patients were informed of the purpose of the study and provided their consent
for the study. The institutional review broad of Shanghai Jiaotong University approved
this study.

Production and purification of the recombinant human SRCR polypeptide

The SRCR domain (residues 421 to 520) of the human MARCO protein was expressed in
Escherichia coli as a six-residue poly-histidine fusion protein by using the pET-28a vector (Novagen,
Darmstadt, Germany). The DNA fragment encoding SRCR was generated by polymerase chain
reaction and the sequence were confirmed by DNA sequencing. The recombined protein
produced in bacteria was purified using Ni-NTA resin (Qiagen, Valencia, CA, USA),
and the purity of sample was determined to be >85% by performing sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The SRCR domain of MARCO was identified by matrix-assisted laser
desorption/ionisation-time of flight mass spectrometry, as described previously [9]. The recombined protein was stored in small aliquots at -80°C.

Anti-class A scavenger receptors autoantibodies assays

For the detection of anti-SRCR and anti-SR-A antibodies, high-binding plates (Costar,
Cambridge, MA, USA) were coated with 10 μg/ml SRCR or 2 μg/ml human SR-AI (R&D Systems,
Inc. Minneapolis, MN, USA) in 0.01 M phosphate-buffered saline (PBS) overnight at
4°C. The plates were washed three times with PBS plus 0.05% Tween 20 and blocked with
PBS containing 0.3% bovine serum albumin for two hours at 37°C. The blocking buffer
was tapped off and washed as described above before the addition of 100 μl of serum
(1:100 diluted in blocking buffer). Serum samples of patients with high titers of
anti-SRCR or anti-SR-A in the pilot enzyme-linked immunosorbent assay (ELISA) were
used as positive controls. The plates with samples were incubated for one hour at
37°C. After washing the plates in the manner described above, the bound human IgG
was detected by horseradish peroxidase-conjugated goat anti-human IgG (Fc specific;
Sigma-Aldrich, St. Louis, MO, USA). After an additional incubation for one hour at
37°C, the plates were washed and 100 μl of the tetramethylbenzidine/H2O2 substrate solution (Kirkegard and Perry Labs, Gaithersburg, MD, USA) was added. The
color development was stopped by the addition of 50 μl of 0.5 M H2SO. Absorbance was measured at a wavelength of 450 nm in a microplate reader (Bio-Rad
Laboratories, Hercules, CA, USA). All samples were run in duplicates and corrected
for background binding. For the anti-SRCR or anti-SR-A antibody, the mean optical
density (OD) value plus three times the standard deviation (SD) of the values determined
for sera collected from healthy controls was used as the cutoff for determining positivity.

IgG purification

IgG were isolated from the sera of patients and healthy donors by using the protein
G affinity column (Pierce, Rockford, IL, USA); the isolates were examined for endotoxin
contamination by using the Limulus amoebocyte lysate kit (A&C Biological Ltd., Zhangjiang,
China). The total protein content was estimated by the BCA kit (Pierce).

Antigen adsorbed IgG

In order to confirm it is the specific activity between the anti-Class A scavenger
receptors antibodies and target antigens that impairs phagocytosis, we carried out
the phagocytosis assay by removal of specific antibodies against SRCR and SR-A. High-biding
plates were coated with antigen and blocked as described above in the ELISA tests.
Purified IgG from each patient (diluted in culturing medium for macrophage) were added
to wells coated with antigens to adsorb anti-SR-A and anti-SRCR antibodies, and the
same samples were also added to wells coated with PBS as controls. Unbound IgG from
antigen-coated and non-antigen-coated wells were collected and incubated with macrophage,
respectively. Phagocytosis tests were performed as described below.

Preparation of monocyte derived macrophages

The human monocytic cell line THP-1 cells (ATCC, Manassas, Va, USA) were cultured
in the same medium as that used for culturing the Jurkat cells. THP-1 were plated
onto 48-well plates at a concentration of 1 × 105 cells/well; 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis,
MO, USA) was then added to the wells and maintained for 24 hours to induce THP-1 cell
differentiation into macrophage. The next day, PMA and the non-adherent cells were
washed off three times with 0.1 M PBS, resulting in adherent macrophages on the plates.
THP-1 derived macrophages by this way were confirmed to express SR-A and MARCO [15,16]. The macrophages were then incubated with fresh medium containing IgG that was purified
from sera with or without anti-class A scavenger receptor antibody for one hour at
37°C in a humidified 5% CO2 incubator.

Phagocytosis assays

In each phagocytosis assay, cells are cultured in the same plate and culture conditions.
After incubation with purified IgG, THP-1-derived macrophages were washed three times
with the culturing medium to remove non-binding IgG, and CFSE-labeled apoptotic and
nonapoptotic Jurkat cells (5 × 105 cells per well) were added to each well to incubate with the macrophages for one hour.
After incubation, the non-binding Jurkat cells were removed by repeated washing with
cold PBS. The remaining firmly adherent cells were trypsinized (0.05% tripsin, Hyclone,
Beijing, China), harvested in PBS/1%BSA, and stained with PERCP-conjugated mouse anti-human
CD3 mAb (BD Pharmingen, SanDiego, CA, USA). Flow cytometry assay was carried out on
an FACSCalibur instrument with CellQuest software (both from BD Bioscience, Mountain
View, CA, USA). Macrophages with ingested apoptotic Jurkat cells were CFSE+CD3-, while macrophages with bound apoptotic Jurkat cells were CFSE+CD3+. Percentages of positive cells were analyzed by using the CellQuest software. The
inhibition effect of IgG on macrophage binding or ingestion of apoptotic cell was
defined as inhibition rate, and the percentages of macrophages binding/ingestion of
apoptotic cells in wells pretreated with PBS rather than IgG were set as 100%. Inhibition
rate (%) = (percentage of binding/ingestion in PBS pretreated well-percentage of binding/ingestion
in IgG pretreated well)/percentage of binding/ingestion in PBS pretreated well × 100.

Statistical analysis

For continuous variables, the comparisons were carried out using the unpaired t-test for two independent samples, the Mann-Whitney U test for non-normal data and paired t-test for matched samples. Categorical variables
between different groups were compared by the chi-square test or Fisher's exact test
when required. Correlations between groups were evaluated by the Spearman test. A
P-value less than 0.05 was considered statistically significant. Data were analyzed
using the SPSS software for Windows (Version 11.0; SPSS Inc, Chicago, IL, USA).

Results

Prevalence of anti-Class A scavenger receptors antibodies

For each individual, the OD values for anti-SRCR and anti-SR-A IgG (Figure 1A, B) were plotted. None of the 85 healthy individuals was anti-SRCR or anti-SR-A positive
(Table 2). Among the 65 patients with SLE, the prevalence of anti-SRCR and anti-SR-A antibody,
as determined by ELISA, was 12 (18.5%) and 22 (33.8%), respectively. The prevalence
of anti-SRCR or anti-SR-A did not show any statistically significant association with
sex or age (data not show). The frequency of anti-SRCR was higher in patients with
SLE than that in patients with RA or pSS (P = 0.005 and P = 0.032, respectively). A higher frequency of anti-SR-A positivity was observed in
SLE patients than in RA or pSS patients (P = 0.007 and P = 0.038, respectively). There was not a statistically significant correlation between
anti-SR-A and anti-SRCR level (P > 0.05).

Table 2. Prevalence of anti-SRCR and anti-SR-A IgG in patients and healthy controls

Figure 1.Levels of anti-class A scavenger receptors IgG in patients and healthy individuals. The optical density (OD) value for each individual is represented as a single point.
Dashed lines indicate the OD values that exceed the mean control value by more than
three standard deviations (SDs). Horizontal bars represent the mean values for each
group. CONTROL, healthy donors; pSS, primary Sjögren syndrome; RA, rheumatoid arthritis;
SLE, systemic lupus erythematosus. A. Levels of anti-SRCR IgG for patients. B. Levels of anti-SR-A IgG for patients.

Inhibition of macrophage uptake of apoptotic cells by anti-class A scavenger receptors
autoantibodies

THP-1-derived macrophages were used to assess the uptake of apoptotic Jurkat cells
in the absence of human serum. Apoptosis of Jurkat cells was confirmed by annexin
V and 7-AAD staining (Figure 2A). CD3 staining was analyzed to discriminate between the binding and ingestion of
apoptotic Jurkat cells (Figure 2B). To test the inhibitory effect of anti-class A scavenger receptor autoantibodies,
we isolated IgG from four SLE patients with anti-SRCR (+) anti-SR-A (+), four SLE
patients with anti-SRCR (+) anti-SR-A(-), four SLE patients with anti-SRCR (-) anti-SR-A
(+), four SLE patients with anti-SRCR (-) anti-SR-A (-) and four healthy controls.
The inhibition rates of phagocytosis for each sample were calculated. IgG with both
anti-SRCR and anti-SR-A antibodies showed higher inhibition rate of ingestion of apoptotic
cells by macrophages than IgG from healthy controls (P < 0.050) (Figure 3A). IgG positive for anti-SRCR or anti-SR-A antibodies showed higher inhibition rates
of ingestion of apoptotic cells by macrophages compared to IgG from normal controls;
however, these differences were not statistically significant (both P > 0.05). The inhibition rates of binding for IgG samples with anti-SR-A or anti-SRCR
antibodies were higher compared to IgG from healthy control (both P < 0.050) (Figure 3B). The inhibition effects of IgG with anti-class A scavenger receptor antibodies (non-antigen
adsorbed) on binding/ingestion of apoptotic cells by macrophage decreased when anti-class
A scavenger receptor antibodies been adsorbed by SRCR and SR-A (antigen adsorbed)
(all P < 0.050) (Figure 3A, B). The decrease in the number of macrophages ingesting apoptotic cells correlated
with the concentration of IgG that were positive for both anti-SRCR and anti-SR-A
(P < 0.050), as shown in Figure 3C.

Figure 2.THP-1 derived macrophage phagocytosis of apoptotic Jurkat cells. A. Apoptosis induction was confirmed by the detection of percentage of Annexin V and
7-AAD staining by flow cytometric assessment. More than 50% cells were Annexin V +7-AAD-(early apoptotic cells) and less than 5% cells were Annexin V +7-AAD+ (late apoptotic and necrotic cells) after ultraviolet B irradiation. This analysis
was repeated prior to each phagocytosis assay. One representative image is shown.
B. Flow cytometry based phagocytosis assay. To discriminate between binding and internalization
of apoptotic Jurkat cells, CD3 staining of CFSE+ macrophages was performed. Macrophages that had ingested apoptotic Jurkat cells were
CD3- (upper left quadrant), while macrophages with Jurkat cells bound to their surface
were CD3+ (upper right quadrant). Macrophages were pretreated with IgG from SLE patients or
healthy controls before being incubated with apoptotic cells, as described in Materials
and Methods. Representative flow cytometry images for phagocytosis by macrophages
pretreated with IgG from control (left panel) and SLE patients with anti-class A scavenger
receptor antibodies (right panel) are shown.

Figure 3.Phagocytosis of apoptotic cells by macrophages that were pretreated with purified
IgG. A, B. Macrophages were pre-treated with IgG from SLE patients who were anti-SR-A positive
(SP), anti-SRCR (anti-MARCO) positive (MP), anti-SR-A and anti-SRCR double-positive
(DP), anti-SRCR and anti-SR-A double negative (DN), or IgG from healthy controls who
are anti-SR-A and anti-SRCR double negative (CONTROL) before being cultured with apoptotic
cells. Purified IgG were incubated with SRCR and SRA (antigen adsorbed) or not (non-antigen
adsorbed) before added to interact with macrophage as described in Materials and Methods.
Pre-incubation with DP reduced the percentage of macrophages ingesting apoptotic cells,
resulted in a significantly higher inhibition rate of ingestion. Pre-incubation with
SP, MP, and DP reduced the percentage of macrophages with apoptotic cells binding
to their surface, as inhibition rates significantly increased. The inhibition rates
significantly decreased when anti-SRCR and anti-SRA antibodies had been adsorbed off
(all P < 0.05). An * indicates a statistically significant difference P < 0.05 by Mann-Whitney U test or paired t-test. Each bar represents the mean + SEM (n = 4). Results are representative of three experiments. C. Dose response of the IgG mediated inhibition of phagocytosis. Decreased percentage
of macrophages ingesting apoptotic cells correlated with increased concentration of
incubating IgG from one SLE patient positive for both anti-SRCR and anti-SR-A (P)
(r = -0.943, P = 0.005). Concentration of IgG from one healthy control (control) did not correlate
with the percentage of macrophages ingesting apoptotic cells (P = 0.208). Bar represents the mean ± SEM (n = 2). Results are representative of three experiments.

Association of anti-class A scavenger receptors autoantibodies with clinical features
of SLE

To investigate the associations between anti-class A scavenger receptors autoantibody
and clinical manifestations or autoantibody profiles, those clinical features were
compared between SLE patients positive and negative for anti-class A scavenger receptors
autoantibodies. The clinical parameters refer to those present at the time of visit
or during the 10 days before presentation, as defined by the SLEDAI-2K criteria. We
identified no significant difference between SLE patients with and those without anti-class
A scavenger receptors antibodies (anti-SRCR or anti-SR-A) in the presence of arthritis,
serositis, mucosal ulcer, neurological or hematological manifestations, lupus nephritis,
and lower levels of C3 or C4 (all P > 0.05). There was no association between anti-class A scavenger receptor autoantibody
(anti-SRCR or anti-SR-A) and any of the autoantibodies such as anti-double-stranded
DNA, anti-Sm, anti-RNP, anti-SSA, anti-SSB, anti-nucleosome, or anti-histone antibodies
(all P > 0.05), either by a qualitative or a quantitative analysis.

Discussion

Increased apoptosis and impaired phagocytic clearance of apoptotic cells could play
important roles in the breakdown of self-tolerance in autoimmune diseases. Phagocytosis
of apoptotic cells by macrophages is a tightly regulated process, which involves a
range of receptors, ligands, and opsonins. Interestingly, some of the ligands and
opsonins that are involved in the interaction between macrophages and apoptotic cells
have been reported to be autoantigens in SLE patients. Reefman et al. [17] demonstrated that autoantibodies recognize antigens expressed on apoptotic cells
and inhibit their uptake via an Fcγ receptor-dependent mechanism. Further, autoantibodies
to serum components acting as bridging molecules (for example, C1q, mannose-binding
lectin, serum amyloid P component, and C-reactive protein) have been detected in patients
with SLE [18,19]. All these findings are indicative of the inhibitory effect of autoantibodies on
the uptake of apoptotic cells by macrophages.

MARCO and SR-A are important binding receptors for apoptotic cells and contribute
to their clearance by macrophages in the splenic marginal zone, an area where blood-borne
apoptotic cells are trapped. Miyake et al. [20] found that the specifically ablation of the macrophages in the marginal zone delayed
clearance of circulating apoptotic cell and impaired immune suppression to apoptotic
cell antigens. With regard to that marginal zone macrophages characterized by high
expression of MARCO and SR-A were responsible for apoptotic cell binding [21], Wermeling and colleagues [8] demonstrated that defects of MARCO and SR-A on these macrophages resulted in antinuclear
autoimmunity. Moreover, Nicola and coworkers [22] observed that the defective expression of MARCO was linked to disease development
via failure of apoptotic cell clearance in the splenic marginal zone in the SLE-prone
mouse strain BXSB. These studies indicted that class A scavenger receptors expressed
on the marginal zone macrophage play an important role in the link between apoptotic
cells clearance and immune tolerance.

It is well known that autoantibodies have been found to precede the symptom onset
of autoimmune diseases for many years in the case of SLE and RA patients [23,24]. Consistent with these observations, autoantibodies against scavenger receptors have
been reported to emerge before the onset of clinical symptoms in SLE-prone mice [8]. Autoantibody against MARCO has been found in the sera of SLE patients [8]. It is intriguing to analyze the influences of anti-class A scavenger receptors antibodies
on the clearance of apoptotic cells and its role in the pathogenesis of the disease.

In this study, autoantibodies to class A scavenger receptors, that is, anti-SRCR and
anti-SR-A, can be found in patients with SLE (18.5% and 33.8%, respectively), RA (3.1%
and 13.8%, respectively), and pSS (0% and 12.0%, respectively). These autoantibodies
are more prevalent in patients with SLE. To explore the effects of anti-class A scavenger
receptors antibodies on the clearance of apoptotic cells by macrophages, THP-1-derived
macrophages that had been confirmed to express MARCO and SR-A were used to study the
interaction between anti-class A scavenger receptors antibodies and macrophages. Our
results indicated that phagocytic clearance of apoptotic cells by macrophages was
inhibited by IgG positive for both anti-SRCR and anti-SR-A in a dose-dependent manner
and partial removal of anti-SRCR and anti-SR-A antibodies decreased the inhibition
effect of those IgG on phagocytosis; this suggests that the blockade of SR-A and the
ligand-binding domain of MARCO by autoantibodies leads to the defective clearance
of apoptotic cells. Treatment with anti-SRCR or anti-SR-A IgG led to significant inhibition
effects on the binding of apoptotic cells by macrophages while only slightly effect
on the ingestion, which may indicate that one of the class A scavenger receptors compensated
for the other blocked one in the uptake of apoptotic cells. Though a wide range of
receptors have been implicated in the uptake of apoptotic cell, including scavenger
receptors of several classes, CD14, CD91, lectins, and the vitronectin receptor [25], expression of individual receptors can increase apoptotic cell adhesion with minimal
effects on uptake [26]. It is possible that decreased binding of apoptotic cells due to impaired MARCO or
SR-A would not reduce the uptake if one of the class A scavenger receptor is qualified.
That can explain this discrepancy in the effect of autoantibodies on the binding and
ingestion of apoptotic cells by macrophages. How these two receptors may interact
to induce apoptotic cell ingestion needs further investigation.

Our study has also found that patients with pSS and RA have anti-class A scavenger
receptors autoantibodies. This finding implies that these antibodies might play a
role in the pathogenesis of autoimmunity. Anti-SR-A and anti-SRCR antibodies were
more prevalent in patients with SLE, which is a disease characteristic of mouse models
of impaired phagocytosis [27]. This suggests that defective phagocytic clearance of apoptotic cells by macrophages
may be more remarkable and critical to triggering a pathologic autoimmune response
in SLE than other autoimmune disease such as pSS or RA.

Conclusions

This study demonstrates that class A scavenger receptors represent new autoantigens
in SLE and other systemic autoimmune disorders. Autoantibodies to class A scavenger
receptors might contribute to the breakdown of self-tolerance by impairing the clearance
of apoptotic debris and play a role in the pathogenesis of autoimmune diseases, especially
SLE. However, only an indirect linkage between these autoantibodies and the breakdown
of self-tolerance has been established, and the function of these antibodies in vivo remains unclear. Additional studies are required to elucidate the complex interaction
between anti-class A scavenger receptor antibodies and macrophage function in animal
models.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

X-WC and YS performed most of the experiments and prepared the manuscript. C-YS worked
on the clinical data presentation and participated in the statistical analysis. C-DY
was responsible for the main experimental design, data interpretation, and for finalizing
the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of
China (No. 30772009; No. 81072471). The authors would like to thank Ms Wei-Zhu Qian
and Mr Xin-Dong Xu for their technical assistance in the production of recombinant
human SRCR polypeptide.